RTEF-1 variants and uses thereof

ABSTRACT

Disclosed are variant RTEF-1 polypeptides having an RTEF-1 amino acid sequence with one or more internal deletions, wherein the polypeptides reduce VEGF promoter activity. Some of the RTEF-1 polypeptides include an amino acid sequence that is at least 80% identical to the contiguous amino acids of 1) amino acids 24 to 47 of SEQ ID NO:15 and 2) each of SEQ ID NOs:16 and 17, but does not comprise the contiguous amino acids of SEQ ID NOs:8, 9, 11, or 12. Also disclosed are nucleic acids encoding the variant RTEF-1 polypeptides of the present invention. Pharmaceutical compositions that include the polypeptides and nucleic acids of the present invention are also disclosed. Methods of inducing cell contact inhibition, regulating organ size, and reducing intracellular YAP activity are also set forth, as well as methods of treating hyperproliferative diseases such as cancer using the pharmaceutical compositions of the present invention.

This application claims the benefit of U.S. Provisional Application No.61/325,675 filed Apr. 19, 2010, which is incorporated herein byreference in its entirety.

The invention was made with government support under Grant No. NEI/NIH1R01EY019042-01A1 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention concerns the fields of molecular biology, cell biology,clinical medicine, pharmacotherapy, and oncology.

2. Description of Related Art

Transcriptional enhancer factor 1-related (RTEF-1) gene is a member ofthe TEA DNA binding domain gene family. The TEA DNA binding domain genefamily is highly conserved from Aspergillus nidulans, yeast, Drosophila,mice to human. The TEA DNA binding family of proteins can be involved inboth activation and repression of different genes and their particularfunction can be modified by association with other proteins (Kaneko &DePamphilis, 1998). Expression of specific members of these genes hasbeen identified in various mammalian tissues, including heart, skeletalmuscle, pancreas, placenta, brain and lung (Stewart et al., 1996;Yasunami et al., 1996; Farrance et al., 1996). Isoforms arising fromalternative splicing of mRNA from a single gene, for transcriptionalenhancer factor-1 (TEF-1) have been identified within a single tissuesuch as the pancreas (Zuzarte et al., 2000; Jiang et al., 2000). Theexpression profile of these genes within the mammalian eye has not beenreported.

Transcripts of the RTEF-1 gene were first identified in chicken tissueand demonstrated to be enriched in cardiac and skeletal muscle (Farranceet al., 1996). The chicken RTEF-1 binds to the myocyte-specific CAT(M-CAT) cis DNA elements and regulates expression of muscle specificgenes, and requires muscle specific cofactors for full transcriptionalactivation. Random screening of 2166 human colorectal cancer cDNAlibrary identified a partial cDNA RTEF-1 sequence which lead to theisolation of a full length human homolog of the avian RTEF-1 from aheart cDNA library (Stewart et al., 1996; Frigerio et al., 1995).Northern blot analysis of human tissue indicated highest levels ofexpression in skeletal muscle and pancreas, with lower levels in heart,kidney and placenta, whereas message was not detected in liver, lung orbrain (Stewart et al., 1996). Northern blot analysis of the mousehomolog of RTEF-1 indicates a different tissue expression pattern whencompared to human. Adult mouse lung tissue expressed the highest level,with very low levels in kidney, heart and skeletal muscle andundetectable amounts in liver, thymus, spleen and brain, whereas RTEF-1message was abundant in mouse embryonic skeletal muscle (Yockey et al.,1996). An alternatively spliced mouse isoform of RTEF-1 that lacks exon5 when compare to the full length gene has been identified in mouseskeletal muscle cells (Yockey et al., 1996).

RTEF-1/TEAD4 has been shown to bind with Yes-associated protein (YAP)and modulate gene expression (Vassilev et al., 2001). Interacting at theend of the Hippo Pathway, TEAD and YAP control organ size duringdevelopment and, thus, play an important role in the coordination ofcell growth, proliferation, and apoptosis (Ota and Sasaki, 2008).Studies have shown that cell to cell interactions elicit signals thoughthe Hippo Pathway which govern YAP-dependent RTEF-1/TEAD4transcriptional activity (Nishioka et al., 2009). Disruption of theHippo Pathway or altered activity of YAP (expression or localization)can lead to cell overgrowth and survival (Zeng and Hong, 2008). The fourhuman TEAD proteins share more than 72% homology. Variable regions arefound in the proline-rich domain and the n-terminus. The n-terminus isthought to be a target for phosphorylation whereas the c-terminus hasbeen shown to bind with YAP. YAP binds to all four TEAD proteins(Vassilev et al., 2001).

YAP is found to be over expressed and diffuse in many tumors. YAPlocation and degree of overexpression varies between cancer types(Steinhardt et al., 2008). Wnt and Akt/PKB are two other pathways thatare tightly regulated during development and seem to play large roles intumor malignancy. Reduction of over expressed YAP activity is thought tobe correlated with decreased cell migration, decreased Akt activation,and increased E-cadherin levels. Studies have shown that 50-80% ofmetastatic cancers express less E-cadherin compared to normal tissues(Orsulic et al., 1999) and YAP overexpression is found in many highlymetastatic cancers that are associated with short survival (Wang et al.,2010). Immunoblotting suggests that E-cadherin and occludin levelsdecrease while N-cadherin, fibronectin, and Akt phosphorylation increasein the presence of YAP (Overholtzer et al., 2006). Likewise, YAP S94Amutation (abolishes YAP and TEAD4 interaction) results in an increase inE-cadherin and gamma-Catenin expression while N-cadherin and fibronectinlevel decrease (Zhao et al., 2008). Some studies have shown that tumorcells will revert to a benign phenotype upon E-cadherinre-establishment. E-cadherin/B-catenin complex is essential for celladhesion. A decrease in E-cadherin results in an increase of freeB-catenin, which can enter the nucleus and activate target genes whichleads to cancer (Wnt pathway) (Semb and Christofori, 1998).

Balancing cell proliferation and apoptosis is essential for propertissue growth, development, and function. Disruption can lead toexcessive tissue loss with subsequence loss of function as in the caseof excessive apoptosis or uncontrolled cell proliferation. The Hippopathway is a potent regulator of tissue homeostasis by controlling cellgrowth, division, and apoptosis. The potent effect of YAP on cellgrowth, division, and apoptosis supports the notion that YAP functionsas to maintain tissue homeostasis. Once dysregulated, it can lead to amalignant phenotype. Malignant cells might produce excess YAP duringgenomic amplification that might overwhelm the normal physiologicregulatory systems and result in abnormal cytoplasmic accumulation.Accumulation of YAP within the cytoplasm maintains a constant pool ofthe protein for nuclear translocation. The stability of YAP may bealtered in neoplastic tissues resulting in ineffective protein turnoverand excessive YAP activity.

Vascular endothelial growth factor (VEGF) is one pro-angiogenic factorthat is known to be up regulated in retinal tissue under hypoxicconditions (Young et al., 1997; Pierce et al., 1996; Donahue et al.,1996; Pe'er et al., 1995). Recently the full length RTEF-1 protein hasbeen identified to not only bind to the VEGF promoter but also toup-regulate the expression of VEGF, for instance under hypoxicconditions in bovine aortic endothelial cells (BAEC) (Shie et al.,2004). Microarray analysis revealed that RTEF-1 expression wasup-regulated by 3-fold in BAEC under hypoxic conditions. Surprisingly,RTEF-1 mediated VEGF gene activation via interaction with Sp 1 elementswithin the VEGF promoter and not M-CAT motifs. In addition RTEF mediatedexpression of VEGF is achieved independently of the hypoxia-induciblefactor (HIF-1) and hypoxia responsive element (HRE) pathway ofactivation (Shie et al., 2004).

VEGF over-expression has been implicated in a variety of angiogenicdisorders such as tumor angiogenesis and aberrant neovascularization.For example, it is well established that VEGF plays an important role inthe development and severity of retinopathy of prematurity (ROP) andother ocular neovascular diseases (Lashkari et al., 2000; Miller, 1997;Vannay et al., 2005; Young et al., 1997). Given the prominent role ofVEGF in such disorders a number of therapeutic strategies for inhibitingVEGF activity have been developed. However, current VEGF blockadetherapies typically involve inhibiting the interaction of extra cellularVEGF with cognate cell surface receptors. Thus, there is a need foralternative strategies for VEGF blockade such as method for inhibitingVEGF production.

SUMMARY OF THE INVENTION

The present invention is in part based on the identification of splicevariants of RTEF-1 that are particularly effective at reducing VEGFproduction. The enhancer isoforms (447 and 1305) are believed to becapable of binding the VEGF promoter to regulate gene expression whereasthe inhibitor isoforms (651, ss651RMR, 366 and 669 and modifications ofthese isoforms), that usually lack a nuclear localization signal,repress VEGF production at the cellular level resulting in lower levelsof secreted VEGF₁₆₅.

Given the prominent role of VEGF in a wide variety of diseases, such ascancer and other diseases associated with vascular proliferation, theseRTEF-1 variants represent a new subset of therapeutic agents. The RTEF-1variants of the present invention are also believed to be effective inreducing YAP activity in cells that overexpress or have elected YAPactivity, such as certain cancer cells. The RTEF-1 variants of thepresent invention can thus be applied in regulating the amount of activeYAP in the cytoplasm or nucleus of target cells, thus regulating cellgrowth and proliferation.

Certain embodiments of the present invention include isolated variantRTEF-1 isoforms that include one or more internal deletions, wherein theisoforms lacks a nuclear translation signal. The result is that theRTEF-1 variant remains confined to the cytoplasm of cells.

Some particular embodiments of the RTEF-1 isoforms of the presentinvention include an amino acid sequence that is at least 80% identicalto the contiguous amino acids of 1) amino acids 24 to 47 of SEQ ID NO:15(the amino acids encoded by exon 10) and 2) each of SEQ ID NO:16 (aminoacids encoded by exon 11) and SEQ ID NO:17 (amino acids encoded by exon12), but does not comprise the contiguous amino acids of SEQ ID NO:8(amino acids encoded by exon 3), SEQ ID NO:9 (amino acids encoded byexon 4), SEQ ID NO:11 (amino acids encoded by exon 6) or SEQ ID NO:12(amino acids encoded by exon 7). The amino acid sequence may be at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to the contiguous amino acidsof 1) amino acids 24 to 47 of SEQ ID NO:15 and 2) each of SEQ ID NO:16and SEQ ID NO:17. In particular embodiments, the variant RTEF-1polypeptide does not include the N-terminal 23 amino acids of SEQ IDNO:15 (which corresponds to the N-terminal 23 amino acids of the aminoacids encoded by exon 10). In particular embodiments, the polypeptidedoes not comprise the contiguous amino acids of SEQ ID NOs: 8, 9, 11,and 12.

In particular embodiments, the isolated variant RTEF-1 polypeptide is atleast 80% identical to “RTEF-1 669” (SEQ ID NO:1) or “RTEF 366” (SEQ IDNO:2). In further embodiments, the RTEF-1 polypeptide is at least 82%,84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO:1 or SEQ ID NO:2. In a particular embodiment, theisolated variant RTEF-1 polypeptide comprises SEQ ID NO:1. In anotherparticular embodiment, the isolated variant RTEF-1 polypeptide comprisesSEQ ID NO:2. In even more specific embodiments, the RTEF-1 polypeptideconsists essentially of SEQ ID NO:1 or SEQ ID NO:2. In further specificembodiments, the RTEF-1 polypeptide consists of SEQ ID NO:1 or SEQ IDNO:2.

In some embodiments, the variant RTEF-1 polypeptide is further definedas a dominant negative RTEF polypeptide (DN RTEF-1 polypeptide). Thismeans that the RTEF-1 variant suppresses or reduces the activity of anintact RTEF-1 polypeptide. For example, in certain aspects, a DN RTEF-1variant may be defined as a polypeptide that when expressed in a cellinhibits or reduces VEGF promoter activity. In particular embodiments,reduction in VEGF promoter activity is reduction relative to the VEGFpromoter activity of a 1305 bp isoform having the sequence set forth inSEQ ID NO:6.

The variant RTEF-1 isoforms may or may not include the N-terminus of ahuman RTEF-1 polypeptide. In some embodiments, the RTEF-1 isoformincludes the first 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25,30, 35, 40, 45, or 50 amino acids of the N-terminus of a human RTEF-1polypeptide. In specific embodiments, the RTEF-1 isoform includes thefirst 9 amino acids of the N-terminus of RTEF-1. In a particularembodiment, the variant RTEF-1 isoform does not include an amino acidsequence consisting of amino acids 1-16 of SEQ ID NO:8.

Particular embodiments of the present variant RTEF-1 isoforms includethe YAP binding domain. One particular variant, SEQ ID NO:1, includesthe YAP binding domain. Another variant, SEQ ID NO:2, differs fromRTEF-1 669 by lacking the first 5 β-sheets and two α-helices of the YAPbinding domain (Chen et al., 2010; Li et al., 2010). Embodiments of thepresent variant RTEF-1 isoforms may harbor a range of YAP bindingaffinities which would be beneficial in modifying YAP activity, such asby suppressing YAP over activity.

In some embodiments of the present variant RTEF-1 polypeptides, thepolypeptide does not include a nuclear localization signal. The variantRTEF-1 polypeptide can then be applied to suppress YAP activity in thecytoplasm of a target cell that may be overexpressing YAP, such as acancer cell. In other embodiments, the variant RTEF-1 polypeptidesinclude a nuclear translocation signal to target suppression of nuclearYAP activity within the nucleus of target cells, such as cancer cellsthat overexpress YAP. Nuclear localization signals are small (5-14)stretches of positively charged amino acids and are usually rich inargenine, lysine, histidine or glycine moieties. Non-limiting examplesof nuclear localization signals include Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val(SEQ ID NO:18) and Arg-Arg-Met-Lys-Trp-Lys-Lys (SEQ ID NO:19).

The variant RTEF-1 polypeptides set forth herein may optionally includea cell internalization moiety. The cell internalization moiety may be apolypeptide, an antibody, an aptamer or an avimer. Non-limiting examplesof cell internalization moieties include a polypeptide from HIV tat,HSV-1 tegument protein VP22, or Drosophila antennopedia. Other examplesinclude poly-arginine, poly-methionine or poly-glycine. In a specificembodiment, the cell internalization moiety includes RMRRMRRMRR (SEQ IDNO:20). In further embodiments, the cell internalization moiety is anantibody that is an IgA, an IgM, an IgE, an IgG, a Fab, a F(ab′)2, asingle chain antibody or a paratope peptide. The variant RTEF-1polypeptides set forth herein may be obtained from natural sources orcan be synthesized using any method known to those of ordinary skill inthe art.

Thus, in certain cases, variant RTEF-1 fusion proteins are providedcomprising a cell internalization moiety (and/or nuclear localizationsequence) and a variant RTEF-1 polypeptide. The skilled artisan withunderstand that such fusion proteins may additionally comprises a one ormore amino acid sequences operating the cell internalizing moiety andthe variant RTEF-1 polypeptide sequence. For example, in some cases alinker sequence may separate these two domains. For example, a linkersequences may comprise a “flexible” amino acids with a large number ordegrees of conformational freedom such as a poly glycine linker In somecases, a linker sequence may comprise a proteinase cleavage site. Forinstance, in certain aspects, a linker sequence may comprising acleavage site that is recognized and cleaved by an intracellularproteinase thereby releasing a variant RTEF-1 sequence from the cellinternalization sequence (or nuclear localization sequence) once thefusion protein has been internalized.

In further aspects of the invention a cell internalization moiety may befurther defined as a cell-targeting moiety, which is a moiety that bindsto or is internalized by only a selected population of cells such ascells expressing a particular cellular receptor. Such a cell targetingmay, for example, comprise an antibody, a growth factor, a hormone, acytokine, an aptamer or an avimer that binds to a cell surface protein.As used herein the term antibody may refer to an IgA, IgM, IgE, IgG, aFab, a F(ab′)2, single chain antibody or paratope peptide. In certaincases, a cell targeting moiety of the invention may target a particulartype of cells such as a retinal, endothelial, iris or neuronal cell. Instill further aspects a cell targeting moiety of the invention may bedefined as cancer cell binding moiety. For example, in some veryspecific cases a cell targeting moiety of the invention may target acancer cell associated antigen such a gp240 or Her-2/neu.

The variant RTEF-1 polypeptide may further include a cell secretionsignal. The cell secretion signal may be any cell secretion signal knownto those of ordinary skill in the art. For example, the cell secretionsignal may include the human IL-2 secretion signal sequence (SEQ IDNO:23). In a particular aspect for example, a variant RTEF-1 polypeptidecomprises a cellular secretion signal. Thus, in certain cases, a variantRTEF-1 polypeptide may comprise a cell internalization moiety and cellsecretion signal, thereby allowing the polypeptide to be secreted by onecells and internalized into a surrounding a cell.

The present invention also includes nucleic acids that include a nucleicacid segment encoding any of the variant RTEF-1 polypeptides of thepresent invention. The nucleic acid may be a DNA or RNA. In someembodiments, the nucleic acid includes an expression cassette thatincludes an antiantiogenesis gene operably coupled to a promoter. Asused herein the term “expression cassette” means that additional nucleicacids sequences are included that enable expression of a variant RTEF-1in a cell, or more particularly in a eukaryotic cell. Such additionalsequences may, for example, comprise a promoter, an enhancer, intronsequences (e.g., before after or with in the variant RTEF-1 codingregion) or a polyadenylation signal sequence. The skilled artisan willrecognize that sequences included in an expression cassette may be usedto alter the expression characteristics of a variant RTEF-1 polypeptide.For instance, cell type specific, conditional or inducible promotersequences may be used to restrict the variant RTEF-1 to selected celltypes or growth conditions. Furthermore, it is contemplated that certainalterations may be made to the variant RTEF-1 polypeptide sequence inorder to enhance expression from an expression cassette for example, asexemplified herein, the initiation codon of a variant RTEF-1 may bechanges to ATG to facilitate efficient translation.

In still further embodiments, the present invention concerns methods forreducing or inhibiting RTEF-1 dependent transcriptional activity. Asused herein the term RTEF-1 dependent transcriptional activity refers totranscription that is mediated or enhanced by expression of an fulllength or fully active RTEF-1 polypeptide, as exemplified by SEQ IDNO:1, or SEQ ID NO:2. Thus, in some respects, the invention providesmethods for inhibiting or reducing VEGF promoter activity (and therebyVEGF expression) comprising expressing a DN RTEF-1 polypeptide in acell. Thus, in a specific embodiment, there is provided a method fortreating a patient with an angiogenic disorder comprising administeringto the patient an effective amount of a therapeutic compositioncomprising a RTEF-1 dominant negative polypeptide or a nucleic acidexpression vector encoding a RTEF-1 dominant negative polypeptide asdescribed supra. In preferred aspects, methods described herein may usedto treat a human patient.

The nucleic acid may optionally be included in a vector. The vector maybe any vector known to those of ordinary skill in the art to targetdelivery of DNA to a particular cell. In particular embodiments, thevector is a viral vector or a liposome. Non-limiting examples of viralvectors include an adenovirus vector, an adeno-associated virus vector,a herpes virus vector, a SV40 virus vector, a retrovirus vector, or avaccinia virus vector. In particular embodiments, the viral vector is aretrovirus vector. In a specific embodiment, the retrovirus vector is alentiviral vector. Examples of lentiviral vectors contemplated as partof the invention include those vectors described in U.S. Pat. No.7,122,181, herein specifically incorporated by reference. The lentiviralexpression vector may optionally be an HIV vector. In some cases suchlentiviral vectors may be self-inactivating (SIN) lentiviral vector suchas those described in U.S. Applns. 20030008374 and 20030082789incorporated herein by reference.

The promoter of the expression cassette may be any promoter known tothose of ordinary skill in the art. For example, the promoter may be acell type specific promoter or an inducible promoter. Non-limitingexamples of inducible promoters include hypoxia inducible promoters andangiogenesis inducible promoters.

The antiangiogenesis gene included in the expression cassette may be anyantiangiogenesis gene known to those of ordinary skill in the art. Forexample, in some aspects, the antiangiogenesis gene may be a tissueinhibitor of metalloproteinase, endostatin, angiostatin, endostatinXVIII, endostatin XV, kringle 1-5, PEX, the C-terminal hemopexin domainof matrix metalloproteinase-2, the kringle 5 domain of humanplasminogen, a fusion protein of endostatin and angiostatin, a fusionprotein of endostatin and the kringle 5 domain of human plasminogen, themonokine-induced by interferon-gamma (Mig), the interferon-alphainducible protein 10 (IP10), a fusion protein of Mig and IP10, solubleFLT-1 (fins-like tyrosine kinase 1 receptor), and kinase insert domainreceptor (KDR) gene. In certain specific aspects, such an angiogenicregulator gene may be delivered in a viral vector such as the lentiviralvectors described in U.S. Pat. No. 7,122,181, incorporated herein byreference. In particular embodiments, the expression cassette includestwo or more antiangiogenesis genes. The two or more antiangiogenesisgenes may be the same or may be distinct genes, such as any of theaforementioned genes.

The present invention also includes pharmaceutical compositions thatinclude one or more variant RTEF-1 polypeptides of the present inventionor one or more nucleic acids of the present invention, and apharmaceutically acceptable carrier. Some of the compositions of thepresent invention may include at least one variant RTEF-1 polyp eptideand at least one nucleic acid encoding a variant RTEF-1 polypeptide. Thepharmaceutical carrier may be any such carrier known to those ofordinary skill in the art. Non-limiting examples of carriers includewater, saline, a dipolar aprotic solvent, an organic solvent, or amixture thereof. The pharmaceutical compositions set forth herein mayoptionally include one or more additional excipients or therapeuticagents. In some embodiments, the pharmaceutical composition is includedin a drug delivery device or coats a drug delivery device. The devicemay be a device designed to be applied to a surface of a subject orwithin a subject. In particular embodiments, the pharmaceuticalcomposition comprises one or more variant RTEF-1 polypeptides thatcomprise SEQ ID NO:1 or SEQ ID NO:2.

Other aspects of the present invention concern methods of inducing cellcontact inhibition in a population that includes two or more cells,comprising contacting a cell of the population with an effective amountof a variant RTEF-1 polypeptide of the present invention or a nucleicacid of the present invention, wherein cell contact inhibition in thepopulation is induced.

Further embodiments of the invention concern methods of regulating sizeof an organ, that involve contacting an organ with an effective amountof a variant RTEF-1 polypeptide of the present invention or a nucleicacid of the present invention, wherein organ size is regulated. Thevariant RTEF-1 polypeptide or nucleic acid may be contacted with theorgan in situ or in vivo.

Still further aspects of the invention concern methods of suppressingYAP activity in a cell that involve contacting a cell with an effectiveamount of a variant RTEF-1 polypeptide of the present invention or anucleic acid of the present invention, wherein YAP activity in the cellis suppressed. The cell may be a cell that demonstrates detectablelevels of YAP in the nucleus and/or the cytoplasm of the cell. The cellmay be a cell that overexpresses YAP in the nucleus or cytoplasm.Non-limiting examples of such cells include cancer cells, such as colonadenocarcinoma cells, lung adenocarcinoma cells, and ovarian serouscystadenocarinoma cells. It has been noted that loss of either criticalcomponents of the Hippo pathway or overexpression of YAP can lead touncontrolled growth and survival. Thus, reduction of YAP activity can bea strategy for reducing uncontrolled growth and prolonging survival insubjects with tumors that overexpress YAP. To target suppression of YAPin the nucleus of a cell, the variant RTEF-1 polypeptide may beengineered to include a nuclear localization sequence. To targetreduction of YAP in the cytoplasm of a cell, the variant RTEF-1polypeptide may be engineered to not include a nuclear localizationsequence. Upon RTEF-1 binding, YAP would be unable to associate with14-3-3 proteins.

Further embodiments of the present invention concern methods of treatinga subject with a disorder that involves administering to the subject aneffective amount of a pharmaceutical composition comprising one or morevariant RTEF-1 polypeptides of the present invention or one or morenucleic acids encoding a variant RTEF-1 polypeptide of the presentinvention, wherein the subject is treated. In some embodiments, thesubject has a disorder associated with abnormal cell growth or abnormalcell proliferation or reduced apoptosis. The disease may be anangiogenic disorder. Non-limiting examples of angiogenic disordersinclude cancer, ocular neovascularization, an arterio-venousmalformation, coronary restenosis, peripheral vessel restenosis,glomerulonephritis, or rheumatoid arthritis. Thus, in certain cases,methods of the invention may be used to treat ocular disorders such asmacular degeneration (e.g., age-related macular degeneration (AMD)),corneal graft rejection, corneal neovascularization, retinopathy ofprematurity (ROP) and diabetic retinopathy (DR). For example, methods ofthe invention may be used in the treatment of wet or dry AMD. Thus, incertain cases, methods of the invention may be used to treat a numberAMD associated ocular lesions such as predominantly classic, minimallyclassic, and occult with no classic lesions (Gragoudas et al., 2004).

In a particular embodiment, the disorder is cancer. Non-limitingexamples of cancer include cancer of breast cancer, lung cancer,prostate cancer, leukemia, lymphoma, head and neck cancer, brain cancer,stomach cancer, intestinal cancer, colorectal cancer, renal cancer,bladder cancer, testicular cancer, esophageal cancer, ocular melanoma,retinoblastoma, liver cancer, ovarian cancer, skin cancer, cancer of thetongue, cancer of the mouth, or metastatic cancer. In some embodiments,the cancer cells demonstrate detectable YAP in the nucleus and/orcytoplasm. In more particular embodiments, the cancer cells demonstratedetectable YAP in the nucleus and the cytoplasm. In further embodiments,the cancer cells demonstrate overexpression of YAP in the nucleus and/orcytoplasm compared to expression of YAP in the nucleus and/or cytoplasmof a cell of a similar tissue type that is not cancerous. For example,the cancer may be a breast cancer cell from a subject, and the cell of asimilar tissue type would be a breast tissue cell of the subject that isnot cancerous. Furthermore additional anticancer therapies may be usedin combination or in conjunction with methods of the invention. Suchadditional therapies may be administered before, after or concomitantlywith methods of the invention. For example an additional anticancertherapy may be a chemotherapy, surgical therapy, an immunotherapy and/orradiation therapy.

In certain particular embodiments, the angiogenic disorder is ocularneovascularization. Non-limiting examples of ocular neovascularizationinclude neovascularization due to age-related macular degeneration,neovascularization due to corneal graft rejection, neovascularizationdue to retinopathy of prematurity (ROP), or neovascularization due todiabetic retinopathy.

The methods of the present invention may further involve administeringto the subject one or more secondary therapies for treatment of adisorder. For example, the secondary therapy may be a secondary therapyof an angiogenic disorder, a disorder associated with abnormal cellgrowth, a disorder associated with abnormal organ growth, or a disorderassociated with impaired cell contact inhibition, or a disorderassociated with increased YAP activity.

In some embodiments, the secondary therapy is an antibody that binds toVEGF, a VEGF receptor, FGF, an FGF receptor, bevacizumab, ranibizumab,or pegaptanib sodium. The secondary therapy may be an anticancer therapythat is chemotherapy, surgical therapy, immunotherapy or radiationtherapy. In particular embodiments, the subject is a mammal.Non-limiting examples of mammals include mice, rats, rabbits, dogs,cats, goats, sheep, horses, cows, primates, and humans. In specificembodiments, the subjects are humans.

Administration of the compositions set forth herein may be by any methodknown to those of ordinary skill in the art. Non-limiting examples ofroutes of administration include intravenously, intraarterially,epidurally, intrathecally, intraperitoneally, subcutaneously, orally, ortopically. In some embodiments directed at the treatment or preventionof an ocular disorder, the composition is administered locally to theeye by topical drops, intracameral injection, subconjunctival injection,subtenon injection, or by intravitreous injection. Further detailconcerning administration and dosage is discussed in the specificationbelow.

Further aspects of the present invention concern kits that include apredetermined quantity of one or more variant RTEF-1 polypeptides of thepresent invention or one or more nucleic acids of the present inventionin one or more sealed vials. The kits may include one or morecomponents, such as vials, syringes, tubes, and instructions for use.

In some further embodiments there is provided a pharmaceuticalcomposition of the invention comprised in a bottle where the bottleincludes an exit portal that enables drop-wise administration of thecomposition. In some cases, a pharmaceutical composition comprised in abottle comprises multiple doses however in certain aspects a bottlecomprises a single dose unit for administration to one or two eyes,preferable a single dose unit is comprised in 1-2 drops of theformulation. As used herein the term “bottle” refers to any fluidcontainer such as an ampoule, dropper or syringe.

Embodiments discussed in the context of a methods and/or composition ofthe invention may be employed with respect to any other method orcomposition described herein. Thus, an embodiment pertaining to onemethod or composition may be applied to other methods and compositionsof the invention as well.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to thedrawings in combination with the detailed description of specificembodiments presented herein.

FIG. 1A, 1B, 1C, 1D. Bar diagrams demonstrating relative VEGF promoteractivity per different variant RTEF-1 isoforms. Bars for hypoxia are onthe right, and bars for normoxia are on the left. 1A—HEK 293 (p7),hypoxia vs. normoxia; 1B—Hek 293 (p7), hypoxia vs. normoxia omitting447; 1C—VEGF promoter activity fold change; 1D—VEGF promoter activityfold change, omitting 447 from the table.

FIG. 2A, 2B, 2C. Schematic of RTEF-1 isoforms. 2A—summary schematic ofisoforms; 2B—1305 pb, 963 bp, 651 bp, and 447 bp isoforms; 2C—669 bp and366 bp isoforms. The gray and black diagonally striped bar indicates theTEA DNA binding domain. The black and white diagonal stripes within theTea Domain represent 3 helices. The grey square shows the position of aputative nuclear localization signal. The vertical striped bar indicatesthe position of the proline rich domain (PRD). The horizontal stripedbars indicate the position of the 2 separate STY domains.

FIG. 3. Schematic showing DNA binding domain and YAP binding domain forRTEF-1 isoforms. The underlined section is the TEA DNA binding domain,and the gray shaded section is the YAP binding domain (SEQ ID NO:3).

FIG. 4. RT-PCR for potential RTEF-1 interacting cofactor proteins withinhuman RVEC, RPE (ARPE-19) and 293T cells.

DETAILED DESCRIPTION OF THE INVENTION

Recently a number of strategies have been developed to inhibitangiogenic signaling. In particular, a number of strategies have focusedon blockade of VEGF signaling by inhibiting the binding of VEGF with oneor both of its cell surface receptors. However, these strategies areunable to address the initial production of VEGF that initiates aberrantangiogenesis. Thus, new methods and compositions that inhibit VEGFproduction may provide new methods for VEGF blockade and treatments forresultant angiogenesis. To this end, in certain aspects, the instantinvention provides variant RTEF-1 polypeptides that inhibit VEGFsignaling. Furthermore, since the instant invention concerns thetargeting of intracellular processes, therapeutics of the invention maybe targeted to specific cell types thereby reducing undesirable systemicside effects. Thus, the instant inventions offers new methods to treatangiogenic disorders and/or ways to enhance the effectiveness of currentVEGF blockade strategies.

RTEF-1 a member of a family of multifunctional transcription factors andhas been shown to be an activator of VEGF transcription, includinghypoxia induced VEGF transcription. The instant invention provides thebasis for new variant RTEF-1 polypeptides and the use thereof to preventor inhibit angiogenic disorders. DN RTEF-1 polypeptides may be delivereddirectly to the intra cellular milieu or expressed in targeted cells toblockade VEGF production. Such dominate negative polypeptides downregulate not only nascent VEGF production but also production of VEGFthat is normally stimulated by RTEF-1 such as during hypoxia. Thus,compositions of the invention, may be used to reduce the ability oftargeted cells and tissues to recruit new blood vessel formation. Thisis of great interest in, for example, in disorders such as cancer andocular neovascular disorders where the production of new blood vesselsis directly related to the pathogenesis of the disease. Furthermore, thevariant RTEF-1 polypeptides may be used to treat tumors or tumormetastases by reducing their ability to gain nutrients through new bloodvessel formation. Thus, methods to slow tumor growth and/or induce tumorregression are also provided. Furthermore, since compositions of theinvention target intracellular transcription, compositions of theinvention may be used to target effected tissues by used of specificcell targeting/internalization moieties and thereby reducing the sideeffects in other non-targeted tissues.

I. Variant RTEF-1 Polypeptides

A number of variant RTEF-1 polypeptides are described and functionallycharacterized herein. For example, particular examples as set forth asfollows:

SEQ ID NO: 1, encoded by a 669 bp RTEF-1 human cDNA that is 222 aminoacid protein, as the following sequence:

MEGTAGTITSKLWMLEFSAFLEQQQDPDTYNKHLFVHIGQSSPSYSDPYLEAVDIRQIYDKFPEKKGGLKDLFERGPSNAFFLVKFWADLNTNIEDEGSSFYGVSSQYESPENMIITCSTKVCSFGKQVVEKVETEYARYENGHYSYRIHRSPLCEYMINFIHKLKHLPEKYMMNSVLENFTILQVVTNRDTQETLLCIAYVFEVSASEHGAQHHIYRLVKE

SEQ ID NO: 2, encoded a 366 bp human cDNA is a 121 amino acid proteinhaving the sequence:

MEGTAGTITPENMIITCSTKVCSFGKQVVEKVETEYARYENGHYSYRIHRSPLCEYMINFIHKLKHLPEKYMMNSVLENFTILQVVTNRDTQETLLCIAYVFEVSASEHGAQHHIYRLV KE

SEQ ID NO: 3, encoded by a 1305 bp human cDNA is a 434 amino acidprotein having the sequence:

MEGTAGTITSNEWSSPTSPEGSTASGGSQALDKPIDNDAEGVWSPDIEQSFQEALAIYPPCGRRKIILSDEGKMYGRNELIARYIKLRTGKTRTRKQVSSHIQVLARRKAREIQAKLKDQAAKDKALQSMAAMSSAQIISATAFHSSMALARGPGRPAVSGFWQGALPGQAGTSHDVKPFSQQTYAVQPPLPLPGFESPAGPAPSPSAPPAPPWQGRSVASSKLWMLEFSAFLEQQQDPDTYNKHLFVHIGQSSPSYSDPYLEAVDIRQIYDKFPEKKGGLKDLFERGPSNAFFLVKFWADLNTNIEDEGSSFYGVSSQYESPENMIITCSTKVCSFGKQVVEKVETEYARYENGHYSYRIHRSPLCEYMINFIHKLKHLPEKYMMNSVLENFTILQVVTNRDTQETLLCIAYVFEVSASEHGAQHHIYRLVKE

SEQ ID NO: 4, encoded by a 936 bp human cDNA is a 311 amino acid proteinhaving the sequence:

MEGTAGTITSNEWSSPTSPEGSTASGGSQALDKPIDNDAEGVWSPDIEQSFQEALAIYPPCGRRKIILSDEGKMYGRNELIARYIKLRTGKTRTRKQVSSHIQVLARRKAREIQAKLKYNKHLFVHIGQSSPSYSDPYLEAVDIRQIYDKFPEKKGGLKDLFERGPSNAFFLVKFWADLNTNIEDEGSSFYGVSSQYESPENMIITCSTKVCSFGKQVVEKVETEYARYENGHYSYRIHRSPLCEYMINFIHKLKHLPEKYMMNSVLENFTILQVVTNRDTQETLLCIAYVFEVSASEHGA QHHIYRLVKE

SEQ ID NO: 5, encoded by a 651 bp human cDNA is a 216 amino acid proteinhaving the sequence:

MEGTAGTITSNEWSSPTSPEGSTASGGSQALDKPIDNDAEGVWSPDIEQSFQEALAIYPPCGRRKIILSDEGKMYGRNELIARYIKLRTGKTSSFYGVSSQYESPENMIITCSTKVCSFGKQVVEKVETEYARYENGHYSYRIHRSPLCEYMINFIHKLKHLPEKYMMNSVLENFTILQVVTNRDTQETLLCIAYVFEVSASEHGAQHHIYRLVKE

SEQ ID NO: 6, encoded by a 447 bp human cDNA is a 148 amino acid proteinhaving the sequence:

MEGTAGTITSNEWSSPTSPEGSTASGGSQALDKPIDNDGEGVWSPDIEQSFQEALAIYPPCGRRKIILSDEGKMYGRNELIARYIKLRTGKTRTRKQVSSHIQVLARRKAREIQAKLKFWQGALPGQAETSHDVKPFSQHHIYRLVKE

As described supra, in certain aspects of the invention a dominantnegative (DN) RTEF-1 polypeptide may comprise one or more internal aminoacid deletions.

TABLE 1  RTEF-1 amino acid sequence by encoding exon ExonAmino acid sequence encoded 1 N/A 2LEGTAGTITSNEWSSPTSPEGSTASGGSQALDKPIDNDAEGVWSPDIEQSFQEALAIYPPCGRRKIILSDEGKMYG* (SEQ ID NO: 7) 3RNELIARYIKLRTGKTRTRKQ (SEQ ID NO: 8) 4VSSHIQVLARRKAREIQAKLK (SEQ ID NO: 9) 5DQAAKDKALQSMAAMSSAQIISATAFHSSMALARGPGRPAVSG (SEQ ID NO: 10) 6FWQGALPGQAGTSHD* (SEQ ID NO: 11) 7 VKPFSQQTYAVQPPLPLPG* (SEQ ID NO: 12)8 FESPAGPAPSPSAPPAPPWQGRSVASSKLWMLEFSAFLEQQQDPDT (SEQ ID NO: 13) 9YNKHLFVHIGQSSPSYSDPYLEAVDIRQIYDKFPEKKGGLKDLFERGPSNAFFLVKFW(SEQ ID NO: 14) 10ADLNTNIEDEGSSFYGVSSQYESPENMIITCSTKVCSFGKQVVEKVE (SEQ ID NO: 15) 11TEYARYENGHYSYRIHRSPLCEYMINFIHKLKHLPEKYMMNSVLENFTILQ (SEQ ID NO: 16) 12VVTNRDTQETLLCIAYVFEVSASEHGAQHHIYRLVKE (SEQ ID NO: 17) *Indicates aminoacids that are encoded by nucleic acid codons that are split betweenexons.

In additional aspects of the invention, variant RTEF polypeptides may befurther modified by one or more amino substitutions while maintainingtheir transcriptional functions. For example, amino acid substitutionscan be made at one or more positions wherein the substitution is for anamino acid having a similar hydrophilicity. The importance of thehydropathic amino acid index in conferring interactive biologic functionon a protein is generally understood in the art (Kyte & Doolittle,1982). It is accepted that the relative hydropathic character of theamino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like. Thus such conservative substitution can be madein an RETF-1 sequence and will likely only have minor effects on theiractivity and ability to repress VEGF promoter activity. As detailed inU.S. Pat. No. 4,554,101, the following hydrophilicity values have beenassigned to amino acid residues: arginine (+3.0); lysine (+3.0);aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine(+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline(−0.5±1); alanine (0.5); histidine −0.5); cysteine (−1.0); methionine(−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine(2.3); phenylalanine (−2.5); tryptophan (−3.4). These values can be usedas a guide and thus substitution of amino acids whose hydrophilicityvalues are within ±2 are preferred, those that are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred. Thus, any of the DN RTEF-1 polypeptides described herein maybe modified by the substitution of an amino acid, for different, buthomologous amino acid with a similar hydrophilicity value. Amino acidswith hydrophilicities within +/−1.0, or +/−0.5 points are consideredhomologous.

Additional information concerning variant RTEF-1 polypeptides can befound in U.S. Patent App. Publ. No. 2009-0117119, herein specificallyincorporated by reference in its entirety.

II. Cell Internalization and Targeting Moieties

Cell internalization moieties for use herein may be any molecule incomplex (covalently or non-covalently) with a variant RTEF-1 thatmediate transport of the variant RTEF-1 across a cell membrane. Suchinternalization moieties may be peptides, polypeptides, hormones, growthfactors, cytokines, aptamers or avimers. Furthermore, cellinternalization moiety may mediate non-specific cell internalization orbe a cell targeting moiety that is internalized in a subpopulation oftargeted cells.

For example, in certain embodiments, cell targeting moieties for use inthe current invention are antibodies. In general the term antibodyincludes, but is not limited to, polyclonal antibodies, monoclonalantibodies, single chain antibodies, humanized antibodies, minibodies,dibodies, tribodies as well as antibody fragments, such as Fab′, Fab,F(ab′)2, single domain antibodies and any mixture thereof. In some casesit is preferred that the cell targeting moiety is a single chainantibody (scFv). In a related embodiment, the cell targeting domain maybe an avimer polypeptide. Therefore, in certain cases the cell targetingconstructs of the invention are fusion proteins comprising a variantRTEF-1 and a scFv or an avimer. In some very specific embodiments thecell targeting construct is a fusion protein comprising variant RTEF-1polypeptide fused to a single chain antibody.

In certain aspects of the invention, a cell targeting moieties may be agrowth factor. For example, transforming growth factor, epidermal growthfactor, insulin-like growth factor, fibroblast growth factor, Blymphocyte stimulator (BLyS), heregulin, platelet-derived growth factor,vascular endothelial growth factor (VEGF), or hypoxia inducible factormay be used as a cell targeting moiety according to the invention. Thesegrowth factors enable the targeting of constructs to cells that expressthe cognate growth factor receptors. For example, VEGF can be used totarget cells that express FLK-1 and/or Flt-1. In still further aspectsthe cell targeting moiety may be a polypeptide BLyS (see U.S. Appln.20060171919).

In further aspects of the invention, a cell targeting moiety may be ahormone. Some examples of hormones for use in the invention include, butare not limited to, human chorionic gonadotropin, gonadotropin releasinghormone, an androgen, an estrogen, thyroid-stimulating hormone,follicle-stimulating hormone, luteinizing hormone, prolactin, growthhormone, adrenocorticotropic hormone, antidiuretic hormone, oxytocin,thyrotropin-releasing hormone, growth hormone releasing hormone,corticotropin-releasing hormone, somatostatin, dopamine, melatonin,thyroxine, calcitonin, parathyroid hormone, glucocorticoids,mineralocorticoids, adrenaline, noradrenaline, progesterone, insulin,glucagon, amylin, erythropoitin, calcitriol, calciferol,atrial-natriuretic peptide, gastrin, secretin, cholecystokinin,neuropeptide Y, ghrelin, PYY3-36, insulin-like growth factor-1, leptin,thrombopoietin, angiotensinogen, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26,IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, or IL-36.As discussed above targeting constructs that comprise a hormone enablemethod of targeting cell populations that comprise extracelluarreceptors for the indicated hormone.

In yet further embodiments of the invention, cell targeting moieties maybe cytokines For example, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9,IL10, IL11, IL12, IL13, IL14, IL15, IL-16, IL-17, IL-18,granulocyte-colony stimulating factor, macrophage-colony stimulatingfactor, granulocyte-macrophage colony stimulating factor, leukemiainhibitory factor, erythropoietin, granulocyte macrophage colonystimulating factor, oncostatin M, leukemia inhibitory factor, IFN-γ,IFN-α, IFN-β, LT-β, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand,4-1BBL, TGF-β, IL 1α, IL-1β, IL-1 RA, MIF and IGIF may all be used astargeting moieties according to the invention.

In certain aspects of the invention a cell targeting moiety of theinvention may be a cancer cell targeting moiety. It is well known thatcertain types of cancer cells aberrantly express surface molecules thatare unique as compared to surrounding tissue. Thus, cell targetingmoieties that bind to these surface molecules enable the targeteddelivery of variant RTEF-1 specifically to the cancers cells. Forexample, a cell targeting moiety may bind to and be internalized by alung, breast, brain, prostate, spleen, pancreatic, cervical, ovarian,head and neck, esophageal, liver, skin, kidney, leukemia, bone,testicular, colon or bladder cancer cell. The skilled artisan willunderstand that the effectiveness of cancer cell targeted variant RTEF-1may, in some cases, be contingent upon the expression or expressionlevel of a particular cancer marker on the cancer cell. Thus, in certainaspects there is provided a method for treating a cancer with targetedvariant RTEF-1 comprising determining whether (or to what extent) thecancer cell expresses a particular cell surface marker and administeringvariant RTEF-1 targeted therapy (or another anticancer therapy) to thecancer cells depending on the expression level of a marker gene orpolypeptide.

As discussed above cell targeting moieties according to the inventionmay be, for example, an antibody. For instance, a cell targeting moietyaccording the invention may bind to a skin cancer cell such as amelanoma cell. It has been demonstrated that the gp240 antigen isexpressed in variety of melanomas but not in normal tissues. Thus, incertain aspects of the invention, there is provided a cell targetingconstruct comprising an variant RTEF-1 and a cell targeting moiety thatbinds to gp240. In some instances, the gp240 binding molecule may be anantibody, such as the ZME-018 (225.28S) antibody or the 9.2.27 antibody.In an even more preferred embodiment, the gp240 binding molecule may bea single chain antibody such as the scFvMEL antibody.

In yet further specific embodiments of the invention, cell targetingconstructs may be directed to breast cancer cells. For example celltargeting moieties that bind to Her-2/neu, such as anti-Her-2/neuantibodies may conjugated to a variant RTEF-1. One example of a suchcell targeting constructs are fusion proteins comprising the singlechain anti-Her-2/neu antibody scFv23 and variant RTEF-1. Other scFvantibodies such as scFv(FRP5) that bind to Her-2/neu may also be used inthe compositions and methods of the current invention (von Minckwitz etal., 2005).

In certain additional embodiments of the invention, it is envisionedthat cancer cell targeting moieties according to invention may have theability to bind to multiple types of cancer cells. For example, the 8H9monoclonal antibody and the single chain antibodies derived therefrombind to a glycoprotein that is expressed on breast cancers, sarcomas andneuroblastomas (Onda et al., 2004). Another example are the celltargeting agents described in U.S. Appln. 2004005647 and in Winthrop etal., 2003 that bind to MUC-1 an antigen that is expressed on a varietycancer types. Thus, it will be understood that in certain embodiments,cell targeting constructs according the invention may be targetedagainst a plurality of cancer or tumor types.

III. Methods for Producing Antibodies

The following methods exemplify some of the most common antibodyproduction methods.

A. Polyclonal Antibodies

Polyclonal antibodies generally are raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the antigen. Asused herein the term “antigen” refers to any polypeptide that will beused in the production of a antibodies. Antigens for use according tothe instant invention include in certain instances, cancer cell surfacemarker polypeptides and eye specific cell surface markers.

It may be useful to conjugate an antigen or a fragment containing thetarget amino acid sequence to a protein that is immunogenic in thespecies to be immunized, e.g. keyhole limpet hemocyanin, serum albumin,bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctionalor derivatizing agent, for example maleimidobenzoyl sulfosuccinimideester (conjugation through cysteine residues), N-hydroxysuccinimide(through lysine residues), glytaraldehyde, succinic anhydride, SOCl₂, orR¹ N═C═NR, where R and R¹ are different alkyl groups.

Animals are immunized against the immunogenic conjugates or derivativesby combining 1 mg of 1 μg of conjugate (for rabbits or mice,respectively) with 3 volumes of Freud's complete adjuvant and injectingthe solution intradermally at multiple sites. One month later theanimals are boosted with ⅕ to 1/10 the original amount of conjugate inFreud's complete adjuvant by subcutaneous injection at multiple sites. 7to 14 days later the animals are bled and the serum is assayed forspecific antibody titer. Animals are boosted until the titer plateaus.Preferably, the animal boosted with the same antigen conjugate, butconjugated to a different protein and/or through a differentcross-linking reagent. Conjugates also can be made in recombinant cellculture as protein fusions. Also, aggregating agents such as alum areused to enhance the immune response.

B. Monoclonal Antibodies

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally-occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

For example, monoclonal antibodies of the invention may be made usingthe hybridoma method first described by Kohler & Milstein (1975), or maybe made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas hamster is immunized as hereinabove described to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the protein used for immunization. Alternatively,lymphocytes may be immunized in vitro. Lymphocytes then are fused withmyeloma cells using a suitable fusing agent, such as polyethyleneglycol, to form a hybridoma cell (Goding 1986).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh level expression of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2cells available from the American Type Culture Collection, Rockville,Md. USA.

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the target antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson & Pollard (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods,Goding (1986). Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium or RPMI-1640 medium. Inaddition, the hybridoma cells may be grown in vivo as ascites tumors inan animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies of the invention is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which are thentransfected into host cells such as simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also may be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences,Morrison et al. (1984), or by covalently joining to the immunoglobulincoding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. In that manner, “chimeric” or “hybrid”antibodies are prepared that have the binding specificity for anyparticular antigen described herein.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody of the invention, or they aresubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibodycomprising one antigen-combining site having specificity for the targetantigen and another antigen-combining site having specificity for adifferent antigen.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

For diagnostic applications, the antibodies of the invention typicallywill be labeled with a detectable moiety. The detectable moiety can beany one which is capable of producing, either directly or indirectly, adetectable signal. For example, the detectable moiety may be aradioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; biotin; radioactive isotopic labels, such as,e.g., ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, or an enzyme, such as alkalinephosphatase, beta-galactosidase or horseradish peroxidase.

Any method known in the art for separately conjugating the antibody tothe detectable moiety may be employed, including those methods describedby Hunter et al. (1962); David et al. (1974); Pain et al. (1981); andNygren (1982).

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays (Zola, 1987).

Competitive binding assays rely on the ability of a labeled standard(which may be a purified target antigen or an immunologically reactiveportion thereof) to compete with the test sample analyte for bindingwith a limited amount of antibody. The amount of antigen in the testsample is inversely proportional to the amount of standard that becomesbound to the antibodies. To facilitate determining the amount ofstandard that becomes bound, the antibodies generally are insolubilizedbefore or after the competition, so that the standard and analyte thatare bound to the antibodies may conveniently be separated from thestandard and analyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insoluble threepart complex. David & Greene, U.S. Pat. No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

C. Humanized Antibodies

As discussed previously, antibodies for use in the methods of theinvention may be polyclonal or monoclonal antibodies or fragmentsthereof. However, in some aspects it is preferred that the antibodiesare humanized such that they do not illict an immune response in subjectbeing treated. Methods for humanizing non-human antibodies are wellknown in the art. Generally, a humanized antibody has one or more aminoacid residues introduced into it from a source which is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the method of Winterand co-workers (Jones et al., 1986); Riechmann et al., 1988; Verhoeyenet al., 1988), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (Cabilly, supra), whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies.

It is important that antibodies be humanized with retention of highaffinity for the antigen and other favorable biological properties, forexample the ability bind to and are internalized by a target cell. Toachieve this goal, according to a preferred method, humanized antibodiesare prepared by a process of analysis of the parental sequences andvarious conceptual humanized products using three dimensional models ofthe parental and humanized sequences. Three dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e. the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from theconsensus and import sequence so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. In general, the CDR residues are directly and mostsubstantially involved in influencing antigen binding. For furtherdetails see U.S. application Ser. No. 07/934,373 filed Aug. 21, 1992,which is a continuation-in-part of application Ser. No. 07/715,272 filedJun. 14, 1991.

D. Human Antibodies

Human monoclonal antibodies can be made by the hybridoma method. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described, for example, by Kozbor(1984) and Brodeur et al. (1987).

It is now possible to produce transgenic animals (e.g. mice) that arecapable, upon immunization, of producing a repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. See, e.g. Jakobovits et al.(1993); Jakobovits et al. (1993).

Alternatively, the phage display technology (McCafferty et al., 1990)can be used to produce human antibodies and antibody fragments in vitro,from immunoglobulin variable (V) domain gene repertoires fromunimmunized donors. According to this technique, antibody V domain genesare cloned in-frame into either a major or minor coat protein gene of afilamentous bacteriophage, such as M13 or fd, and displayed asfunctional antibody fragments on the surface of the phage particle.

Because the filamentous particle contains a single-stranded DNA copy ofthe phage genome, selections based on the functional properties of theantibody also result in selection of the gene encoding the antibodyexhibiting those properties. Thus, the phage mimicks some of theproperties of the B-cell. Phage display can be performed in a variety offormats; for their review see, e.g. Johnson et al. (1993). Severalsources of V-gene segments can be used for phage display. Clackson etal. (1991) isolated a diverse array of anti-oxazolone antibodies from asmall random combinatorial library of V genes derived from the spleensof immunized mice. A repertoire of V genes from unimmunized human donorscan be constructed and antibodies to a diverse array of antigens(including self-antigens) can be isolated essentially following thetechniques described by Marks et al. (1991), or Griffith et al. (1993).In a natural immune response, antibody genes accumulate mutations at ahigh rate (somatic hypermutation). Some of the changes introduced willconfer higher affinity, and B cells displaying high-affinity surfaceimmunoglobulin are preferentially replicated and differentiated duringsubsequent antigen challenge. This natural process can be mimicked byemploying the technique known as “chain shuffling” (Marks et al., 1992).In this method, the affinity of “primary” human antibodies obtained byphage display can be improved by sequentially replacing the heavy andlight chain V region genes with repertoires of naturally occurringvariants (repertoires) of V domain genes obtained from unimmunizeddonors. This technique allows the production of antibodies and antibodyfragments with affinities in the nM range. A strategy for making verylarge phage antibody repertoires (also known as “the mother-of-alllibraries”) has been described by Waterhouse et al. (1993), and theisolation of a high affinity human antibody directly from such largephage library has been reported. Gene shuffling can also be used toderive human antibodies from rodent antibodies, where the human antibodyhas similar affinities and specificities to the starting rodentantibody. According to this method, which is also referred to as“epitope imprinting”, the heavy or light chain V domain gene of rodentantibodies obtained by phage display technique is replaced with arepertoire of human V domain genes, creating rodent-human chimeras.Selection on antigen results in isolation of human variable capable ofrestoring a functional antigen-binding site, i.e. the epitope governs(imprints) the choice of partner. When the process is repeated in orderto replace the remaining rodent V domain, a human antibody is obtained(see PCT patent application WO 93/06213, published Apr. 1, 1993). Unliketraditional humanization of rodent antibodies by CDR grafting, thistechnique provides completely human antibodies, which have no frameworkor CDR residues of rodent origin.

IV. Nucleic Acid Molecules

In certain aspects, the instant invention concerns nucleic acidmolecules encoding a variant RTEF-1 polypeptide as set forth herein. Incertain aspects, a nucleic acid sequence is comprised in a nucleic acidvector. The term “vector” is used to refer to a carrier nucleic acidmolecule into which a nucleic acid sequence can be inserted forintroduction into a cell where it can be replicated. A nucleic acidsequence can be “exogenous,” which means that it is foreign to the cellinto which the vector is being introduced or that the sequence ishomologous to a sequence in the cell but in a position within the hostcell nucleic acid in which the sequence is ordinarily not found. Vectorsinclude plasmids, cosmids, viruses (bacteriophage, animal viruses, andplant viruses), and artificial chromosomes (e.g., YACs). One of skill inthe art would be well equipped to construct a vector through standardrecombinant techniques (see, for example, Maniatis et al., 1988 andAusubel et al., 1994, both incorporated herein by reference).

The term “expression vector” refers to any type of genetic constructcomprising a nucleic acid coding for RNA capable of being transcribed.In some cases, RNA molecules are then translated into a protein,polypeptide, or peptide. In other cases, these sequences are nottranslated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host cell. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

A. Promoters and Enhancers

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind, such as RNA polymerase and other transcriptionfactors, to initiate the specific transcription a nucleic acid sequence.The phrases “operatively positioned,” “operatively linked,” “undercontrol,” and “under transcriptional control” mean that a promoter is ina correct functional location and/or orientation in relation to anucleic acid sequence to control transcriptional initiation and/orexpression of that sequence.

A promoter generally comprises a sequence that functions to position thestart site for RNA synthesis. The best known example of this is the TATAbox, but in some promoters lacking a TATA box, such as, for example, thepromoter for the mammalian terminal deoxynucleotidyl transferase geneand the promoter for the SV40 late genes, a discrete element overlyingthe start site itself helps to fix the place of initiation. Additionalpromoter elements regulate the frequency of transcriptional initiation.Typically, these are located in the region 30 110 bp upstream of thestart site, although a number of promoters have been shown to containfunctional elements downstream of the start site as well. To bring acoding sequence “under the control of” a promoter, one positions the 5′end of the transcription initiation site of the transcriptional readingframe “downstream” of (i.e., 3′ of) the chosen promoter. The “upstream”promoter stimulates transcription of the DNA and promotes expression ofthe encoded RNA.

The spacing between promoter elements frequently is flexible, so thatpromoter function is preserved when elements are inverted or movedrelative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either cooperatively or independently to activatetranscription. A promoter may or may not be used in conjunction with an“enhancer,” which refers to a cis-acting regulatory sequence involved inthe transcriptional activation of a nucleic acid sequence.

A promoter may be one naturally associated with a nucleic acid sequence,as may be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other virus, or prokaryotic or eukaryotic cell, andpromoters or enhancers not “naturally occurring,” i.e., containingdifferent elements of different transcriptional regulatory regions,and/or mutations that alter expression. For example, promoters that aremost commonly used in recombinant DNA construction include the βlactamase (penicillinase), lactose and tryptophan (trp) promotersystems. In addition to producing nucleic acid sequences of promotersand enhancers synthetically, sequences may be produced using recombinantcloning and/or nucleic acid amplification technology, including PCR™, inconnection with the compositions disclosed herein (see U.S. Pat. Nos.4,683,202 and 5,928,906, each incorporated herein by reference).Furthermore, it is contemplated the control sequences that directtranscription and/or expression of sequences within non-nuclearorganelles such as mitochondria, chloroplasts, and the like, can beemployed as well.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in theorganelle, cell type, tissue, organ, or organism chosen for expression.Those of skill in the art of molecular biology generally know the use ofpromoters, enhancers, and cell type combinations for protein expression,(see, for example Sambrook et al. 1989, incorporated herein byreference). The promoters employed may be constitutive, tissue-specific,inducible, and/or useful under the appropriate conditions to direct highlevel expression of the introduced DNA segment, such as is advantageousin the large-scale production of recombinant proteins and/or peptides.The promoter may be heterologous or endogenous.

Additionally any promoter/enhancer combination (as per, for example, theEukaryotic Promoter Data Base EPDB, http://www.epd.isb-sib.ch/) couldalso be used to drive expression. Use of a T3, T7 or SP6 cytoplasmicexpression system is another possible embodiment. Eukaryotic cells cansupport cytoplasmic transcription from certain bacterial promoters ifthe appropriate bacterial polymerase is provided, either as part of thedelivery complex or as an additional genetic expression construct.

The identity of tissue-specific promoters or elements, as well as assaysto characterize their activity, is well known to those of skill in theart. Nonlimiting examples of such regions include the human LIMK2 gene(Nomoto et al. 1999), the somatostatin receptor 2 gene (Kraus et. al.,1998), murine epididymal retinoic acid-binding gene (Lareyre et. al.,1999), human CD4 (Zhao-Emonet et. al., 1998), mouse alpha2 (XI) collagen(Tsumaki, et. al., 1998), DIA dopamine receptor gene (Lee, et. al.,1997), insulin-like growth factor II (Wu et. al., 1997), and humanplatelet endothelial cell adhesion molecule-1 (Almendro et. al., 1996).

B. Initiation Signals and Internal Ribosome Binding Sites

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements, such as a Kozak sequence, which is comprised of the followingsequence and spans the initiation codon AUG (ACCaugG).

In certain embodiments of the invention, the use of internal ribosomeentry sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′ methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picornavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES element from a mammalian message (Macejak andSarnow, 1991). IRES elements can be linked to heterologous open readingframes. Multiple open reading frames can be transcribed together, eachseparated by an IRES element, creating polycistronic messages. By virtueof the IRES element, each open reading frame is accessible to ribosomesfor efficient translation. Multiple genes can be efficiently expressedusing a single promoter/enhancer to transcribe a single message (seeU.S. Pat. Nos. 5,925,565 and 5,935,819, each herein incorporated byreference).

C. Multiple Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleicacid region that contains multiple restriction enzyme sites, any ofwhich can be used in conjunction with standard recombinant technology todigest the vector (see, for example, Carbonelli et. al., 1999, Levensonet. al., 1998, and Cocea, 1997, incorporated herein by reference.)“Restriction enzyme digestion” refers to catalytic cleavage of a nucleicacid molecule with an enzyme that functions only at specific locationsin a nucleic acid molecule. Many of these restriction enzymes arecommercially available. Use of such enzymes is widely understood bythose of skill in the art. Frequently, a vector is linearized orfragmented using a restriction enzyme that cuts within the MCS to enableexogenous sequences to be ligated to the vector. “Ligation” refers tothe process of forming phosphodiester bonds between two nucleic acidfragments, which may or may not be contiguous with each other.Techniques involving restriction enzymes and ligation reactions are wellknown to those of skill in the art of recombinant technology.

D. Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing toremove introns from the primary transcripts. Vectors containing genomiceukaryotic sequences may require donor and/or acceptor splicing sites toensure proper processing of the transcript for protein expression (see,for example, Chandler et. al., 1997, herein incorporated by reference.)

E. Termination Signals

The vectors or constructs of the present invention will generallycomprise at least one termination signal. A “termination signal” or“terminator” is comprised of the DNA sequences involved in specifictermination of an RNA transcript by an RNA polymerase and these include;UGA, UAA and UAG. Thus, in certain embodiments a termination signal thatends the production of an RNA transcript is contemplated. A terminatormay be necessary in vivo to achieve desirable message levels.

In eukaryotic systems, the terminator region may also comprise specificDNA sequences that permit site-specific cleavage of the new transcriptso as to expose a polyadenylation site (AAUAAA). This signals aspecialized endogenous polymerase to add a stretch of about 200 Aresidues (polyA) to the 3′ end of the transcript. RNA molecules modifiedwith this polyA tail appear to more stable and are translated moreefficiently. Thus, in other embodiments involving eukaryotes, it ispreferred that that terminator comprises a signal for the cleavage ofthe RNA, and it is more preferred that the terminator signal promotespolyadenylation of the message. The terminator and/or polyadenylationsite elements can serve to enhance message levels and to minimize readthrough from the cassette into other sequences. In certain specificcases a polyadenylation signal may be the signal from neuropilin-1 asdescribed in U.S. Appln. 20050175591.

Terminators contemplated for use in the invention include any knownterminator of transcription described herein or known to one of ordinaryskill in the art, including but not limited to, for example, thetermination sequences of genes, such as for example the bovine growthhormone terminator or viral termination sequences, such as for examplethe SV40 terminator. In certain embodiments, the termination signal maybe a lack of transcribable or translatable sequence, such as due to asequence truncation.

F. Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively an autonomously replicating sequence (ARS) can be employedif the host cell is yeast.

G. Selectable and Screenable Markers

In certain embodiments of the invention, cells containing a nucleic acidconstruct of the present invention may be identified in vitro or in vivoby including a marker in the expression vector. Such markers wouldconfer an identifiable change to the cell permitting easy identificationof cells containing the expression vector. Generally, a selectablemarker is one that confers a property that allows for selection. Apositive selectable marker is one in which the presence of the markerallows for its selection, while a negative selectable marker is one inwhich its presence prevents its selection. An example of a positiveselectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with FACS analysis. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable and screenable markers are well known to one of skill in theart.

H. Plasmid Vectors

In certain embodiments, a plasmid vector is contemplated for use totransform a host cell. In general, plasmid vectors containing repliconand control sequences which are derived from species compatible with thehost cell are used in connection with these hosts. The vector ordinarilycarries a replication site, as well as marking sequences which arecapable of providing phenotypic selection in transformed cells. In anon-limiting example, E. coli is often transformed using derivatives ofpBR322, a plasmid derived from an E. coli species. pBR322 contains genesfor ampicillin and tetracycline resistance and thus provides easy meansfor identifying transformed cells. The pBR plasmid, or other microbialplasmid or phage must also contain, or be modified to contain, forexample, promoters which can be used by the microbial organism forexpression of its own proteins.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example, thephage lambda GEMTM 11 may be utilized in making a recombinant phagevector which can be used to transform host cells, such as, for example,E. coli LE392.

Further useful plasmid vectors include pIN vectors (Inouye et. al.,1985); and pGEX vectors, for use in generating glutathione S transferase(GST) soluble fusion proteins for later purification and separation orcleavage. Other suitable fusion proteins are those with β galactosidase,ubiquitin, and the like.

Bacterial host cells, for example, E. coli, comprising the expressionvector, are grown in any of a number of suitable media, for example, LB.The expression of the recombinant protein in certain vectors may beinduced, as would be understood by those of skill in the art, bycontacting a host cell with an agent specific for certain promoters,e.g., by adding IPTG to the media or by switching incubation to a highertemperature. After culturing the bacteria for a further period,generally of between 2 and 24 h, the cells are collected bycentrifugation and washed to remove residual media.

I. Viral Vectors

The ability of certain viruses to infect cells or enter cells viareceptor mediated endocytosis, and to integrate into host cell genomeand express viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign nucleic acids into cells (e.g.,mammalian cells). DN RTEF-1 components of the present invention may be aviral vector that encodes a DN RTEF-1 pollypeptide. Non-limitingexamples of virus vectors that may be used to deliver a nucleic acid ofthe present invention are described below.

1. Adenoviral Vectors

A particular method for delivery of the nucleic acid involves the use ofan adenovirus expression vector. Although adenovirus vectors are knownto have a low capacity for integration into genomic DNA, this feature iscounterbalanced by the high efficiency of gene transfer afforded bythese vectors. “Adenovirus expression vector” is meant to include thoseconstructs containing adenovirus sequences sufficient to (a) supportpackaging of the construct and (b) to ultimately express a tissue orcell specific construct that has been cloned therein. Knowledge of thegenetic organization or adenovirus, a 36 kb, linear, double stranded DNAvirus, allows substitution of large pieces of adenoviral DNA withforeign sequences up to 7 kb (Grunhaus and Horwitz, 1992).

2. AAV Vectors

The nucleic acid may be introduced into the cell using adenovirusassisted transfection. Increased transfection efficiencies have beenreported in cell systems using adenovirus coupled systems (Kelleher andVos, 1994; Cotten et. al., 1992; Curiel, 1994). Adeno associated virus(AAV) is an attractive vector system for use in the delivery of DNRTEF-1 expression cassettes of the present invention as it has a highfrequency of integration and it can infect nondividing cells, thusmaking it useful for delivery of genes into mammalian cells, forexample, in tissue culture (Muzyczka, 1992) or in vivo. AAV has a broadhost range for infectivity (Tratschin et. al., 1984; Laughlin et. al.,1986; Lebkowski et. al., 1988; McLaughlin et. al., 1988). Detailsconcerning the generation and use of rAAV vectors are described in U.S.Pat. Nos. 5,139,941 and 4,797,368, each incorporated herein byreference.

3. Retroviral Vectors

Retroviruses have promise as DN RTEF-1 delivery vectors in therapeuticsdue to their ability to integrate their genes into the host genome,transferring a large amount of foreign genetic material, infecting abroad spectrum of species and cell types and of being packaged inspecial cell lines (Miller, 1992).

In order to construct a DN RTEF-1 retroviral vector, a nucleic acid(e.g., one encoding a DN RTEF-1) is inserted into the viral genome inthe place of certain viral sequences to produce a virus that isreplication defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et. al., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into a special cell line (e.g., bycalcium phosphate precipitation for example), the packaging sequenceallows the RNA transcript of the recombinant plasmid to be packaged intoviral particles, which are then secreted into the culture media (Nicolasand Rubenstein, 1988; Temin, 1986; Mann et. al., 1983). The mediacontaining the recombinant retroviruses is then collected, optionallyconcentrated, and used for gene transfer. Retroviral vectors are able toinfect a broad variety of cell types. However, integration and stableexpression require the division of host cells (Paskind et. al., 1975).

Lentiviruses are complex retroviruses, which, in addition to the commonretroviral genes gag, pol, and env, contain other genes with regulatoryor structural function. Methods for delivery of antiangiogenic moleculeswith lentiviral vectors have been previously described, see for exampleU.S. Pat. No. 7,122,181, U.S. Patent App. Publ. Nos. 20090148936,20060062765, 20030082159, and 20020114783, each of which is incorporatedherein by reference in its entirety. Lentiviral vectors are well knownin the art (see, for example, Naldini et. al., 1996; Zufferey et. al.,1997; Blomer et. al., 1997; U.S. Pat. Nos. 6,013,516 and 5,994,136).Some examples of lentivirus include the Human Immunodeficiency Viruses:HIV-1, HIV-2 and the Simian Immunodeficiency Virus: SIV. Lentiviralvectors have been generated by multiply attenuating the HIV virulencegenes, for example, the genes env, vif, vpr, vpu and nef are deletedmaking the vector biologically safe.

Recombinant lentiviral vectors are capable of infecting non-dividingcells and can be used for both in vivo and ex vivo gene transfer andexpression of nucleic acid sequences. For example, recombinantlentivirus capable of infecting a non-dividing cell wherein a suitablehost cell is transfected with two or more vectors carrying the packagingfunctions, namely gag, pol and env, as well as rev and tat is describedin U.S. Pat. No. 5,994,136, incorporated herein by reference. One maytarget the recombinant virus by linkage of the envelope protein with anantibody or a particular ligand for targeting to a receptor of aparticular cell-type. By inserting a sequence (including a regulatoryregion) of interest into the viral vector, along with another gene whichencodes the ligand for a receptor on a specific target cell, forexample, the vector is now target-specific.

4. Other Viral Vectors

Other viral vectors may be employed as vaccine constructs in the presentinvention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et. al., 1988),sindbis virus, cytomegalovirus and herpes simplex virus may be employed.They offer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et.al., 1988; Horwich et. al., 1990).

5. Delivery Using Modified Viruses

A nucleic acid to be delivered may be housed within an infective virusthat has been engineered to express a specific binding ligand. The virusparticle will thus bind specifically to the cognate receptors of thetarget cell and deliver the contents to the cell. A novel approachdesigned to allow specific targeting of retrovirus vectors was developedbased on the chemical modification of a retrovirus by the chemicaladdition of lactose residues to the viral envelope. This modificationcan permit the specific infection of hepatocytes via sialoglycoproteinreceptors.

Another approach to targeting of recombinant retroviruses was designedin which biotinylated antibodies against a retroviral envelope proteinand against a specific cell receptor were used. The antibodies werecoupled via the biotin components by using streptavidin (Roux et. al.,1989). Using antibodies against major histocompatibility complex class Iand class II antigens, they demonstrated the infection of a variety ofhuman cells that bore those surface antigens with an ecotropic virus invitro (Roux et. al., 1989).

J. Vector Delivery and Cell Transformation

Suitable methods for nucleic acid delivery for transformation of anorganelle, a cell, a tissue or an organism for use with the currentinvention are believed to include virtually any method by which anucleic acid (e.g., DNA) can be introduced into an organelle, a cell, atissue or an organism, as described herein or as would be known to oneof ordinary skill in the art. Such methods include, but are not limitedto, direct delivery of DNA such as by ex vivo transfection (Wilson et.al., 1989, Nabel et al, 1989), by injection (U.S. Pat. Nos. 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610,5,589,466 and 5,580,859, each incorporated herein by reference),including microinjection (Harland and Weintraub, 1985; U.S. Pat. No.5,789,215, incorporated herein by reference); by electroporation (U.S.Pat. No. 5,384,253, incorporated herein by reference; Tur-Kaspa et. al.,1986; Potter et. al., 1984); by calcium phosphate precipitation (Grahamand Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et. al., 1990); byusing DEAE dextran followed by polyethylene glycol (Gopal, 1985); bydirect sonic loading (Fechheimer et. al., 1987); by liposome mediatedtransfection (Nicolau and Sene, 1982; Fraley et. al., 1979; Nicolau et.al., 1987; Wong et. al., 1980; Kaneda et. al., 1989; Kato et. al., 1991)and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988);by microprojectile bombardment (PCT Application Nos. WO 94/09699 and95/06128; U.S. Pat. Nos. 5,610,042; 5,322,783 5,563,055, 5,550,318,5,538,877 and 5,538,880, and each incorporated herein by reference); byagitation with silicon carbide fibers (Kaeppler et. al., 1990; U.S. Pat.Nos. 5,302,523 and 5,464,765, each incorporated herein by reference); byAgrobacterium mediated transformation (U.S. Pat. Nos. 5,591,616 and5,563,055, each incorporated herein by reference); by PEG mediatedtransformation of protoplasts (Omirulleh et. al., 1993; U.S. Pat. Nos.4,684,611 and 4,952,500, each incorporated herein by reference); bydesiccation/inhibition mediated DNA uptake (Potrykus et. al., 1985), andany combination of such methods. Through the application of techniquessuch as these, organelle(s), cell(s), tissue(s) or organism(s) may bestably or transiently transformed.

V. Therapeutic Methods

A. Pharmaceutical Preparations

Therapeutic compositions for use in methods of the invention may beformulated into a pharmacologically acceptable format. The phrases“pharmaceutical or pharmacologically acceptable” refers to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, such as, forexample, a human, as appropriate. The preparation of an pharmaceuticalcomposition that contains at least one variant RTEF-1 polypeptide ornucleic acid active ingredient will be known to those of skill in theart in light of the present disclosure, as exemplified by Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990,incorporated herein by reference. Moreover, for animal (e.g., human)administration, it will be understood that preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed., 1990, incorporated herein by reference). A pharmaceuticallyacceptable carrier is preferably formulated for administration to ahuman, although in certain embodiments it may be desirable to use apharmaceutically acceptable carrier that is formulated foradministration to a non-human animal, such as a canine, but which wouldnot be acceptable (e.g., due to governmental regulations) foradministration to a human. Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the therapeutic orpharmaceutical compositions is contemplated.

The actual dosage amount of a composition of the present inventionadministered to a subject can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. In other non-limitingexamples, a dose may also comprise from about 1 microgram/kg/bodyweight, about 5 microgram/kg/body weight, about 10 microgram/kg/bodyweight, about 50 microgram/kg/body weight, about 100 microgram/kg/bodyweight, about 200 microgram/kg/body weight, about 350 microgram/kg/bodyweight, about 500 microgram/kg/body weight, about 1 milligram/kg/bodyweight, about 5 milligram/kg/body weight, about 10 milligram/kg/bodyweight, about 50 milligram/kg/body weight, about 100 milligram/kg/bodyweight, about 200 milligram/kg/body weight, about 350 milligram/kg/bodyweight, about 500 milligram/kg/body weight, to about 1000 mg/kg/bodyweight or more per administration, and any range derivable therein. Innon-limiting examples of a derivable range from the numbers listedherein, a range of about 5 mg/kg/body weight to about 100 mg/kg/bodyweight, about 5 microgram/kg/body weight to about 500 milligram/kg/bodyweight, etc., can be administered, based on the numbers described above.

In particular embodiments, the compositions of the present invention aresuitable for application to mammalian eyes. For example, the formulationmay be a solution, a suspension, or a gel. In some embodiments, thecomposition is administered via a bioerodible implant, such as anintravitreal implant or an ocular insert, such as an ocular insertdesigned for placement against a conjunctival surface. In someembodiments, the therapeutic agent coats a medical device or implantabledevice.

In preferred aspects the formulation of the invention will be applied tothe eye in aqueous solution in the form of drops. These drops may bedelivered from a single dose ampoule which may preferably be sterile andthus rendering bacteriostatic components of the formulation unnecessary.Alternatively, the drops may be delivered from a multi-dose bottle whichmay preferably comprise a device which extracts preservative from theformulation as it is delivered, such devices being known in the art.

In other aspects, components of the invention may be delivered to theeye as a concentrated gel or similar vehicle which forms dissolvableinserts that are placed beneath the eyelids.

Furthermore, the therapeutic compositions of the present invention maybe administered in the form of injectable compositions either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid prior to injection may also be prepared. Thesepreparations also may be emulsified. A typical composition for suchpurpose comprises a pharmaceutically acceptable carrier. For instance,the composition may contain 10 mg, 25 mg, 50 mg or up to about 100 mg ofhuman serum albumin per milliliter of phosphate buffered saline. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike.

Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oil and injectable organic esters such as ethyloleate.Aqueous carriers include water, alcoholic/aqueous solutions, salinesolutions, parenteral vehicles such as sodium chloride, Ringer'sdextrose, etc. Intravenous vehicles include fluid and nutrientreplenishers. Preservatives include antimicrobial agents, anti-oxidants,chelating agents and inert gases. The pH and exact concentration of thevarious components the pharmaceutical composition are adjusted accordingto well known parameters.

Additional formulations are suitable for oral administration. Oralformulations include such typical excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate and the like. Thecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders. When the route istopical, the form may be a cream, ointment, salve or spray.

An effective amount of the therapeutic composition is determined basedon the intended goal. The term “unit dose” or “dosage” refers tophysically discrete units suitable for use in a subject, each unitcontaining a predetermined-quantity of the therapeutic compositioncalculated to produce the desired responses, discussed above, inassociation with its administration, i.e., the appropriate route andtreatment regimen. The quantity to be administered, both according tonumber of treatments and unit dose, depends on the protection desired.Thus, in some case dosages can be determined by measuring for examplechanges in serum insulin or glucose levels of a subject.

Precise amounts of the therapeutic composition may also depend on thejudgment of the practitioner and are peculiar to each individual.Factors affecting the dose include the physical and clinical state ofthe patient, the route of administration, the intended goal of treatment(e.g., alleviation of symptoms versus attaining a particular seruminsulin or glucose concentration) and the potency, stability andtoxicity of the particular therapeutic substance.

In particular embodiments, the compositions of the present invention aresuitable for application to mammalian eyes. For example, the formulationmay be a solution, a suspension, or a gel. In some embodiments, thecomposition is administered via a bioerodible implant, such as anintravitreal implant or an ocular insert, such as an ocular insertdesigned for placement against a conjunctival surface. In someembodiments, the therapeutic agent coats a medical device or implantabledevice.

In preferred aspects the formulation of the invention will be applied tothe eye in aqueous solution in the form of drops. These drops may bedelivered from a single dose ampoule which may preferably be sterile andthus rendering bacteriostatic components of the formulation unnecessary.Alternatively, the drops may be delivered from a multi-dose bottle whichmay preferably comprise a device which extracts preservative from theformulation as it is delivered, such devices being known in the art.

In other aspects, components of the invention may be delivered to theeye as a concentrated gel or similar vehicle which forms dissolvableinserts that are placed beneath the eyelids.

B. Additional Therapies

As discussed supra in certain aspects therapeutic methods of theinvention may be used in combination or in conjunction with additionalantiangiogenic or anticancer therapies.

1. Chemotherapy

In certain embodiments of the invention DN RTEF-1 is administered inconjunction with a chemo therapeutic agent. For example, cisplatin(CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide,camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea,dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin,mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptorbinding agents, taxol, paclitaxel, gemcitabien, navelbine,farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil,vincristin, Velcade, vinblastin and methotrexate, or any analog orderivative variant of the foregoing may used in methods according to theinvention.

2. Radiotherapy

In certain further embodiments of the invention, compositions of theinvention may be used to sensitize cell to radiation therapy. Radiotherapy may include, for example, γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. In certain instancesmicrowaves and/or UV-irradiation may also used according to methods ofthe invention. Dosage ranges for X-rays range from daily doses of 50 to200 roentgens for prolonged periods of time (3 to 4 wk), to single dosesof 2000 to 6510 roentgens. Dosage ranges for radioisotopes vary widely,and depend on the half-life of the isotope, the strength and type ofradiation emitted, and the uptake by the neoplastic cells.

The terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a therapeutic construct and achemotherapeutic or radio therapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing or stasis, both agents are delivered to a cell in acombined amount effective to kill the cell or prevent it from dividing.

3. Immunotherapy

Immunotherapeutics, generally, rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The immune effectormay be, for example, an antibody specific for some marker on the surfaceof a tumor cell. The antibody alone may serve as an effector of therapyor it may recruit other cells to actually effect cell killing. Theantibody also may be conjugated to a drug or toxin (chemotherapeutic,radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) andserve merely as a targeting agent. Alternatively, the effector may be alymphocyte carrying a surface molecule that interacts, either directlyor indirectly, with a tumor cell target. Various effector cells includecytotoxic T cells and NK cells.

Immunotherapy, thus, could be used as part of a combined therapy, inconjunction with gene therapy. The general approach for combined therapyis discussed below. Generally, the tumor cell must bear some marker thatis amenable to targeting, i.e., is not present on the majority of othercells. Many tumor markers exist and any of these may be suitable fortargeting in the context of the present invention. Common tumor markersinclude carcinoembryonic antigen, prostate specific antigen, urinarytumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72,HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, lamininreceptor, erb B, Her-2/neu, gp240 and p155.

4. Genes

In yet another embodiment, gene therapy in which a therapeuticpolynucleotide is administered before, after, or at the same time as acell targeting construct of the present invention. Delivery of variantRTEF-1 in conjunction with a vector encoding one or more additional geneproducts may have a combined anti-hyperproliferative effect on targettissues. A variety of genes are encompassed within the invention, forexample a gene encoding p53 may be delivered in conjunction with variantRTEF-1 compositions.

5. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery is a cancer treatment that may beused in conjunction with other therapies, such as the treatment of thepresent invention, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies. A variant RTEF-1therapy of the invention may be employed alone or in combination with acytotoxic therapy as neoadjuvant surgical therapy, such as to reducetumor size prior to resection, or it may be employed as postadjuvantsurgical therapy, such as to sterilize a surgical bed following removalof part or all of a tumor.

Curative surgery includes resection in which all or part of canceroustissue is physically removed, excised, and/or destroyed. Tumor resectionrefers to physical removal of at least part of a tumor. In addition totumor resection, treatment by surgery includes laser surgery,cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs'surgery). It is further contemplated that the present invention may beused in conjunction with removal of superficial cancers, precancers, orincidental amounts of normal tissue.

Upon excision of part of all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

6. Other Agents

Hormonal therapy may also be used in conjunction with the presentinvention or in combination with any other cancer therapy previouslydescribed. The use of hormones may be employed in the treatment ofcertain cancers such as breast, prostate, ovarian, or cervical cancer tolower the level or block the effects of certain hormones such astestosterone or estrogen. This treatment is often used in combinationwith at least one other cancer therapy as a treatment option or toreduce the risk of metastases.

EXAMPLES

The following examples are included to further illustrate variousaspects of the invention. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples that followrepresent techniques and/or compositions discovered by the inventor tofunction well in the practice of the invention, and thus can beconsidered to constitute preferred modes for its practice. However,those of skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentswhich are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

Example 1 Relative VEGF Promoter Activity of Variant RTEF-1 Isoforms

Studies were conducted to examine the relative VEGF promoter activity ofselected isoforms of RTEF.

The pSEAP assay is essentially a chemiluminescence assay in which twoplasmids are electroporated into cells. One plasmid (pcDNA) contains aspecific RTEF-1 isoform and the other plasmid (pSEAP plasmid) containsthe VEGF promoter fused to the secreted alkaline phosphatase gene.Transcriptional activity on the exogenous VEGF promoter results in asecretable, thermo-stable, alkaline phosphatase (AP) protein. Theabundance of AP protein can be measured by a luminescene assay. Thus,the measured lumens are indicative of the amount of AP in the media,which is a directly correlated to the activity on the promoter (humanVEGF) driving the expression of the AP gene. Lumens measured from the‘no insert’ control are indicative of the endogenous transcriptionfactors (and cofactors) acting upon the exogenous VEGF promoter.

Cells were grown to approximately 80% confluency. Cells weretrypsinized, washed with 8 mls of serum containing media and centrifugedat 1000 rpm for 5 minutes. The media was removed and cells wereresuspended in an appropriate volume of solution V transfection reagent(AMAXA). One hundred microliters of the cell mix was added toelectroporation cuvettes (AMAXA) containing 2 μg of a pcDNA RTEF-1isoform expression vector along with 2 μg of the pSEAP expression vectorunder the control of the human VEGF promoter either containing orlacking the HRE element. Cells were transfected with the electroporationapparatus (AMAXA) using a predetermined optimized transfection program.Cells were then plated into either 6-well or 24-well plates andincubated overnight in 3 mls or 1 ml of appropriate media respectively.The following day the media was changed to 2% serum using a minimalvolume to completely cover the cells. Cells were divided into either anatmospheric oxygen environment (normoxic) or a 1% oxygen environment(hypoxic). Cells were incubated in these states for 24-48 hours.Following the time course, cell media was collected and used to performa secretable alkaline phosphate assay following manufactures protocols(Clontech). Briefly, 25 μl of media was added to 75 μl of dilutionbuffer, plated into 96 well plate and incubated at 65° C. for 30minutes. The plate was then cooled on ice for 3 minutes and allowed toequilibrate to room temperature. 100 μl of pSEAP substrate solution wasadded to each well and incubated away from light for 15-30 minutes.Plates were then read within 60 minutes using plate reader capable ofdetecting luminescence.

RTEF-1 isoforms effect the VEGF promoter differently. Cells arecotransfected (Amaxa Nucleofector machine program AO23), plated, andallowed to recover in 10% serum media overnight at 37° C. and 5% CO₂.The media is changed to that of 2% serum the next morning. One of theduplicate trays are placed in a hypoxic chamber and flushed for 8 min.at 20 liters/min with 1% O₂, 5% CO₂, and 94% N₂. The hypoxic chamber isthen sealed. Both the hypoxic chamber and the ‘normoxic’ tray are placedinto the incubator (37° C. with 5% CO₂). The hypoxic chamber is flushedas previously done after 7 hrs and both trays are placed into theincubator overnight. The next morning, the hypoxic chamber is flushedonce more as noted above and place into the incubator. Media iscollected 2-3 hours later. Time in hypoxic chamber is ˜27-48 hrs.

TABLE 2 1305 669 651 447 366 No insert Normoxic Lumens Avg. of Avgs.108185.37 4440.60 27590.20 308097.60 7835.20 34289.00 SD 24234.071251.01 12294.25 30271.97 137.65 1859.00 Hypoxic Lumens Avg. of Avgs.203921.27 8937.67 33275.07 453160.20 11343.00 42251.27 SD 16329.02698.28 5937.96 91022.17 1472.96 4959.10 Lumen Fold Change Normoxic 3.160.13 0.80 8.99 0.23 1.00 Hypoxic 4.83 0.21 0.79 10.73 0.27 1.00

Results are shown in FIG. 1A-1D. The fold changes for each isoform arelisted in Table 2. RTEF-1 isoform 1305 and 447 stimulate activity of theVEGF promoter; whereas RTEF-1 isoforms 669, 651, and 366 display aninhibitory effect.

Note that lumens measured in the no insert control (pcDNA plasmid w/oRTEF-1+pSEAP plasmid) are due to the endogenous activity on theexogenous VEGF promoter. Thus, any cotransfections with RTEF-1 isoformsthat result in measured lumens below this value are believed to be theresult of RTEF-1 isoforms interacting with endogenous cofactorsnecessary for VEGF transcriptional activity.

Results

FIG. 2A-2C depict a schematic diagram of various RTEF isoforms. The 669bp variant RTEF-1 and the 366 bp variant RTEF-1 isoform are depicted inFIG. 2E. As can be seen in FIG. 1, the 669 bp variant RTEF-1 and the 366bp variant RTEF-1 isoform demonstrated significantly reduced VEGFpromoter activity under hypoxic and normoxic conditions compared toother RTEF-1 isoforms that were tested. A p-value of less than 0.05 isindicative of a statistically significant difference.

In contrast to the RTEF-1 1305, 936, 651, and 447 isoforms that wereharvested from retinal endothelial cells, RTEF-1 366 and 669 isoformswere isolated from Mel 202 cells (human choroidal melanoma cells).

Under hypoxic conditions, all RTEF-1 isoforms are significantlydifferent from the no insert control except for the 651 isoform. RTEF-1isoforms 366 and 669 show no significant difference (see Table 3).

TABLE 3 Unpaired T-Test p - values: Measured Lumens between RTEF-1isoforms under Hypoxic Conditions RTEF-1 No Isoform 1305 669 651 447 366insert 1305  669 0.0023 651 0.0011 0.0182 447 0.0381 0.0137 0.0150 3660.0022 0.0877 0.0187 0.0138 No insert 0.0017 0.0065 0.1171 0.0157 0.0052

Under normoxic conditions, all isoforms are significantly different fromthe no insert control except for the 651 isoform. RTEF-1 isoform 651shows no significant difference from that of the 366 or 669 isoforms.This is thought to be due to the higher variation between triplicatesamples in the 651 normoxic wells (see Table 4).

TABLE 4 Unpaired T-Test p - values: Measured Lumens between RTEF-1isoforms under Normoxic Conditions RTEF-1 No Isoform 1305 669 651 447366 insert 1305  669 0.0175 651 0.0147 0.0811 447 0.0011 0.0033 0.0013366 0.0189 0.0410 0.1085 0.0034 No insert 0.0334 0.0001 0.4455 0.00390.0016

When comparing RTEF-1 isoforms under hypoxic to normoxic conditions,RTEF-1 651, 447, 366, and the no insert control showed significantdifference (Table 5).

TABLE 5 Measured Lumens between hypoxic and normoxic conditions for eachRTEF-1 isoform RTEF-1 No Isoform 1305 669 651 447 366 insert P - value0.0070 0.0109 0.5248 0.0980 0.0529 0.0946

Example 2 Expression of SRF, MAX, and YAP65 in Cell Lines

Materials and Methods

Hek293 cells were grown in 6-well plates with appropriate media to 80%confluency. The media was removed and the cells were washed with PBS.Cellular RNA was isolated using the RNAquous-4 PCR kit (Ambion®).Briefly, 300 μl of lysis buffer was added to the cells and incubated for5 minutes at room temperature. The lysate was collected and an equalvolume of 65% ethanol was added and mixed by inversion. The mixture waspassed through an RNA collection filter by centrifugation at 12,000 rpmand washed three times with wash buffer (Ambion®) by centrifugation at12,000 rpm. Filters were centrifuged once more to completely dry thefilter. Elution buffer (Ambion®) heated to 75° C. was added to thecolumn and centrifuged to elute the RNA. RNA was quantified by a UVspectrophotometer using a 260 nm wavelength. Total RNA at aconcentration of 250 ng was used for reverse transcription using theOmni-script RT kit (Qiagen). The reaction was composed of 2 μl of 10× RTbuffer, 2 μl of dNTP mix (5 mM each dNTP), 2 μl of oligo-dT primer (10μM), 1 μl of RNAse inhibitor (10 U/μl), 1 μl of reverse transcriptaseand water. Reactions were incubated at 37° C. for one hour. Theresulting cDNA was then used for PCR. The reaction was composed of 2 μlof the cDNA mixed with 2.5 μl of 10× buffer, 0.2 mM dNTPs, 20 pmols ofthe forward primer (5′tgtttcagccgcagcctctc3′; SEQ ID NO:21) and 20 pmolsof reverse primer (5′tgggggctgcttcactgg3′; SEQ ID NO:22), 1 μl ofRedTaq® and water. Reactions were incubated at 95° C. for 5 minutes with35 cycles of 95° C. for 25 seconds, 55° C. for 20 seconds and 72° C. for45 seconds, then 72° C. for 5 minutes. The PCR reactions were run out ona 1.5% agarose gel, visualized with ethidium bromide and photographedusing an integrating camera.

Results

Results are shown in FIG. 4. All cell lines tested showed expression ofSRF (lanes 1, 5 and 10) and Max (lanes 2, 6 and 11) and YAP65 (lanes 4,8 and 13). The TAZ message is not amplifiable from RPE cells (lane 7)but is present in RVEC (lane 3) and 293T cells (lane 13).

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. An isolated variant RTEF-1 polypeptide thatreduces VEGF promoter activity comprising an amino acid sequence that isat least 95% identical to the contiguous amino acids of 1) amino acids24 to 47 of SEQ ID NO:15 and 2) each of SEQ ID NOs:16 and 17, but doesnot comprise the contiguous amino acids of SEQ ID NOs:8, 9, 11, and 12,further wherein said polypeptide does not include an amino acid sequenceconsisting of amino acids 1-16 of SEQ ID NO:8.
 2. The isolated variantRTEF-1 polypeptide of claim 1, comprising an amino acid sequence that isat least 97% identical to the contiguous amino acids of 1) amino acids24 to 47 of SEQ ID NO:15 and 2) each of SEQ ID NOs:16 and
 17. 3. Theisolated variant RTEF-1 polypeptide of claim 1, comprising an amino acidsequence that is at least 99% identical to the contiguous amino acidsof 1) amino acids 24 to 47 of SEQ ID NO:15 and 2) each of SEQ ID NOs:16and
 17. 4. The isolated variant RTEF-1 polypeptide of claim 1, whereinthe polypeptide is at least 95% identical to SEQ ID NO:1.
 5. Theisolated RTEF-1 polypeptide of claim 4, wherein the polypeptide is atleast 97% identical to SEQ ID NO:1.
 6. The isolated RTEF-1 polypeptideof claim 5, wherein the polypeptide is at least 99% identical to SEQ IDNO:1.
 7. The isolated RTEF-1 polypeptide of claim 6, wherein thepolypeptide comprises SEQ ID NO:1.
 8. The isolated RTEF-1 polypeptide ofclaim 1, wherein the polypeptide is at least 95% identical to SEQ IDNO:2.
 9. The isolated RTEF-1 polypeptide of claim 8, wherein thepolypeptide is at least 97% identical to SEQ ID NO:2.
 10. The isolatedRTEF-1 polypeptide of claim 8, wherein the polypeptide is at least 99%identical to SEQ ID NO:2.
 11. The isolated RTEF-1 polypeptide of claim10, wherein the RTEF-1 polypeptide comprises SEQ ID NO:2.
 12. Theisolated RTEF-1 polypeptide of claim 1, further comprising a cellinternalization moiety or a nuclear localization sequence that is apolypeptide, an antibody, an aptamer or an avimer.
 13. The isolatedRTEF-1 polypeptide of claim 12, wherein the cell internalization moietyis a polypeptide comprising an HIV tat polypeptide, HSV-1 tegumentprotein VP22, or Drosophila antennopedia protein.
 14. The isolatedRTEF-1 polypeptide of claim 12, wherein the cell internalization moietyis a polypeptide comprising poly-arginine, poly-methionine orpoly-glycine.
 15. The isolated RTEF-1 polypeptide of claim 12, whereinthe cell internalization moiety is a polypeptide comprising RMRRMRRMRR(SEQ ID NO:20).
 16. The isolated RTEF-1 polypeptide of claim 12, whereinthe cell internalization moiety is an antibody that is an IgA, an IgM,an IgE, an IgG, a Fab, a F(ab′)2, a single chain antibody or a paratopepeptide.
 17. The isolated RTEF-1 polypeptide of claim 1, furthercomprising a cell secretion signal.
 18. The isolated RTEF-1 polypeptideof claim 17, wherein the cell secretion signal comprises the human IL-2secretion signal sequence (SEQ ID NO:23).
 19. A kit comprising apredetermined quantity of a variant RTEF-1 polypeptide of claim 1 in oneor more sealed vials.
 20. A pharmaceutical composition comprising a) oneor more variant RTEF-1 polypeptides as set forth in claim 1; and b) apharmaceutically acceptable carrier.
 21. The pharmaceutical compositionof claim 20, comprising SEQ ID NO:1 or SEQ ID NO:2.
 22. A method ofinducing cell contact inhibition in a population comprising two or morecells, comprising contacting a cell of the population with an effectiveamount of a variant RTEF-1 polypeptide of claim 1, wherein cell contactinhibition in the population is induced.
 23. A method of suppressing YAPactivity in a cell, comprising contacting a cell with an effectiveamount of a variant RTEF-1 polypeptide of claim 1, wherein YAP activityin the cell is suppressed.
 24. The method of claim 23, wherein the cellis contacted with a variant RTEF-1 polypeptide comprising a nuclearlocalization sequence and activity of YAP in the nucleus of the cell issuppressed.
 25. The method of claim 23, wherein the variant RTEF-1polypeptide does not include a nuclear localization sequence and whereintranslocation of YAP into the nucleus of the cell is inhibited.
 26. Amethod for treating a subject with a disorder associated with abnormalcell growth or abnormal cell proliferation, comprising administering toa subject with a disorder associated with abnormal cell growth orabnormal cell proliferation an effective amount of a pharmaceuticalcomposition comprising a variant RTEF-1 polypeptide according to claim1: wherein the disorder is treated.
 27. The method of claim 26, whereinthe disorder associated with abnormal cell growth or abnormal cellproliferation is an angiogenic disorder that is cancer, ocularneovascularization, an arterio-venous malformation, coronary restenosis,peripheral vessel restenosis, glomerulonephritis, or rheumatoidarthritis.
 28. The method of claim 26, wherein the angiogenic disorderis cancer.
 29. The method of claim 28, wherein the cancer is a cancerselected from the group consisting of cancer of breast cancer, lungcancer, prostate cancer, leukemia, lymphoma, head and neck cancer, braincancer, stomach cancer, intestinal cancer, colorectal cancer, renalcancer, bladder cancer, testicular cancer, esophageal cancer, ocularmelanoma, retinoblastoma, liver cancer, ovarian cancer, skin cancer,cancer of the tongue, cancer of the mouth, or metastatic cancer.
 30. Themethod of claim 28, wherein the cancer is comprised of cancer cells thatdemonstrate detectable YAP in the nucleus and/or cytoplasm.
 31. Themethod of claim 28, wherein the cancer cells demonstrate overexpressionof YAP in the nucleus and/or cytoplasm compared to expression of YAP inthe nucleus and/or cytoplasm of a cell of a similar tissue type that isnot cancerous.
 32. The method of claim 27, wherein the angiogenicdisorder is ocular neovascularization.
 33. The method of claim 32,wherein the ocular neovascularization is neovascularization due toage-related macular degeneration, neovascularization due to cornealgraft rejection, neovascularization due to retinopathy of prematurity(ROP), or neovascularization due to diabetic retinopathy.
 34. The methodof claim 27, further comprising administering a secondary therapy fortreatment of the antiangiogenic disorder.
 35. The method of claim 34,wherein the secondary therapy is an antibody that binds to VEGF, a VEGFreceptor, FGF, an FGF receptor, bevacizumab, ranibizumab, or pegaptanibsodium.
 36. The method of 34, wherein the secondary therapy is ananticancer therapy that is chemotherapy, surgical therapy, immunotherapyor radiation therapy.
 37. The method of claim 26, wherein the subject isa human.
 38. The method of claim 26, wherein the composition isadministered intravenously, intraarterially, epidurally, intrathecally,intraperitoneally, subcutaneously, orally, or topically.
 39. The methodof claim 32, wherein the composition is administered locally to the eyeby topical drops, intracameral injection, subconjunctival injection,subtenon injection, or by intravitreous injection.